Microscopes are employed in a wide variety of fields to view various types of biological and non-biological samples. In some applications, for example in certain applications of life sciences, confocal microscopes are preferred over more traditional wide-field microscopes. In a confocal microscope, light is passed through a small aperture (traditionally a pinhole) positioned at an optically conjugate plane. The point illumination substantially eliminates out-of-focus light (background signal) and thereby increases the optical resolution and contrast of the image acquired. However, these advantages are realized at the expense of decreased light intensity caused by the confocal aperture, such that longer exposure times are often required in comparison to wide-field microscopes.
As only a single small point on the sample is illuminated at any time, the confocal microscope often provides a scanning function. One type of scanning technique entails the use of a spinning disk, also known as a scanning disk or Nipkow disk. A typical spinning disk includes multiple apertures arranged along one or more spiral paths. Scanning is implemented by spinning the disk at a high angular velocity, for example thousands of revolutions per minute (RPM).
Two problems associated with microscopes, including confocal microscopes such as spinning disk microscopes, are reflectance of stray light from optical components and autofluorescence exhibited by optical components (i.e., generation of secondary stray light in an optical component). Reflected stray light includes light that is not part of the image of the sample and, in the case of fluorescence microscopy, light that is not part of the fluorescent response of the sample. Autofluorescence in optical components is generated by color centers in such optical components. The color centers are due to the presence of rare earth elements and other impurities in the material (e.g., optical glass or polymer) of the optical components. Optical components that may exhibit unacceptably high reflectance and autofluorescence include the spinning disk as well as lenses and windows positioned on the optical axis of the microscope.
Conventionally, these problems have been addressed by coating such optical components with a conventional material specified as being a low-reflectance material or an anti-reflective material. Additionally, the microscope may include an emission filter effective for blocking a large portion of unwanted light. Nevertheless, even when such measures are taken an unacceptable amount of stray light and autofluorescence-based light may reach the imaging device of the microscope. This “ghost” light has the appearance of background in confocal images, thus lowering signal-to-background ratio for a given exposure time or increasing the exposure time required to attain a desired signal-to-background ratio. Therefore, it would be desirable to reduce the amount of stray light reflected from optical components and light emitted from optical components due to autofluorescence.